Patient
The cell line was derived from a liver metastasis of colon cancer patient who was a 69-year-old woman in Jiangsu province hospital. 2014 the patient was carried out the surgery of colon cancer. In 2016 Dec, laboratory examination results showed CEA 64.4 ng/ml, CA199 24.4 U/ml, CA724 29.9 U/ml, NSE 32.4 ng/ml; Colonoscopy biopsy showed Sigmoid adenocarcinoma. MRI examination demonstrated a space-occupying lesion in the right lobe. The resected liver tumor was approximately 5 × 5 × 2.5 cm, pathological results showed hepatic adenocarcinoma, II–III stage.
Establishment of the HCS1220 cell line
A portion of tumor tissue (3 gm) resected from liver during the operation and immediately washed in DMEM medium. The tissue was cut 1 mm3 into tissue-culture flasks (Corning Glass Works, Corning, NY), washed twice with l × PBS (Ca2+, Mg2+ free) and 1 × Wash Medium (Invitrogen), After digestion using 0.1% Collagenase Type IV (Gibco) in DMEM for 30 min at 37 °C and shaked occasionally in a 15 ml centrifuge tube (Corning), the suspension was filtered by 45 μm cell strainer to remove large fragments and the collected liquid was centrifuged consecutively at 1500 rpm, 1000 rpm, 800 rpm and 600 rpm for 5 min, respectively. Cancer cells were resuspended using primary culture medium (DMEM/F12 + 10% FBS + 1% penicillin–streptomycin + 0.2 U/ml insulin) and transferred to a tissue-culture flask overnight in a humidified incubator at 37 °C with 5% CO2. Fresh medium can be changed every day.
In the first 3 months, fibroblast cells grew fast. During this time, some tumor cell “island” emerged. We removed the fibroblast scratching by pipetting tips and the tumor cell “island” became bigger. Tumor cell clones were picked out from the primary culture and purified, at passage 40 (13 months after we picked out the tumor cell clone), a stable cell line (HCS1220) was considered to have been established.
Morphological of HCS1220
HCS1220 were seeded in 25-mm3 tissue-culture flasks and incubated at 37 °C in a humidified atmosphere containing 5% CO2 for 2 weeks. Everyday the cells were placed under the inverted microscope to observe the general morphology.
DNA isolation and STR (short tandem repeat) analysis
Genomic DNA from HCS1220 was isolated using genomic extraction kit (Axygen, USA). The cell DNA was amplified by a 20-STR amplification method. STR loci and the sex gene Amelogenin were tested by an ABI 3730XL Genetic Analyzer. The data were analyzed by GeneScan and GeneMapperTM ID Software (Invitrogen).
Karyotype analysis
We performed chromosomal analysis on HCS1220 cells with low and high passages to determine whether chromosomal abnormalities could be considered in vivo or in vitro phenomena. The cells in logarithmic growth phase were harvested by trypsinization and DMEM/F12 medium were added to stop the reaction. After centrifugation at 1000 rpm for 4 min, 0.56% KCl solution (37 °C) was added and the hypotonic treatment was performed for 30 min. Then the fixative (methanol/acetic acid 3:l) was added to fix the cells twice at room temperature for 30 min. Slides were air-dried and stained with Giemsa stain for 20 min.
Transplantation
HCS1220 cells were digested with 0.25% trypsin and then counted. After centrifugation at 950 rpm for 3 min, the supernatant was discarded. The cells were resuspend in PBS to prepare a cell suspension of 1 × 106 cells/ml.
Four 6-week-old BALB/c nude mice (Slac Laboratory Animal Company. Ltd, Shanghai) were subcutaneous injected 0.1 ml suspension into the groin and the tumor formation was observed from the 7th day. The mice were sacrificed when tumor burden exceed 10% of the normal body weight. The viable tumor tissues were fixed in 4% formaldehyde, paraffin embedded and diagnosed (H&E staining).
Detection of biomarkers for xenografts
The expression of biomarkers CK20 and CDX2 for colon cancer and biomarkers AFP, hep-1 and glypican-3 for liver cancer were determined by immunocytochemistry assay. After deparaffinization and antigen retrieval procedure, slides were permeabilized with 0.1% Triton X-100, incubated 5–10 min at room temperature with 0.3% H2O2 solution and washed with distilled water and soak in PBS twice. Then blocked with 5–10% goat serum (Invitrogen, USA) in PBS at room temperature, after 10 min, The slides were incubated with the following primary antibodies at room temperature overnight: anti-CK20, anti-CDX2, anti-AFP, anti-hep-1 and anti-glypican-3. The secondary antibodies were supplied at 37 °C for 10–30 min. Next horseradish enzyme or alkaline phosphatase labeled streptavidin working solution could be added to incubate at 37 °C for 10–30 min. Finally, the hematoxylin was used in counterstaining.
Detection of mycoplasma contamination
Myco-PCR-Mix mycophenolate mycoplasma detection kit (Qiao Xinzhou Bioengineering Co. Ltd.) was used to detect mycoplasma contamination.
